The overall objective of this grant proposal is to characterize the receptor-binding domain- and the receptor-binding site within this domain - of diphtheria toxin, as well as to characterize fully the specific diphtheria toxin-binding cell surface protein(s) from highly toxin-sensitive Vero cells that function as the toxin receptor. The receptor-binding domain has recently been localized to the carboxyl- terminal Mr about 6,000 region of diphtheria toxin by our laboratory. The receptor-binding site will be localized further by chemical modifications o specific amino acid residues and by site- directed mutagenesis. Monoclonal antibodies to the toxin's receptor-binding domain/site will be prepared and will be employed to produce anti- idiotypic antibodies; the anti-idiotypic antibodies will then be tested for their ability to protect Vero Cells from diphtheria toxin-mediated cytotoxicity and for their ability to recognize the toxin receptor. Antibodies to the receptor will be employed to characterize and purify the receptor, and to investigate the cell surface distribution and mechanism of internalization of the toxin receptor on Vero cells. The diphtheria toxin-sensitive mouse cells, recently isolated by transfection of toxin-resistant mouse L-cells with DNA from Vero cells, will be employed to clone and sequence the toxin receptor; the sequence will be compared to that of other receptors, in order to determine the nature of the toxin receptor and/or its homology with other known receptors. The results of this proposed project will provide an insight into the possible nature of the physiological cell surface receptor that diphtheria toxin utilizes to gain illicit access to the cell cytosol, will significantly extend our knowledge on toxin:receptor interactions, and will further our understanding on receptor-mediated internalization of such macromolecules as growth factors, hormones, and other exotoxins.